During corticogenesis, excitatory neurons are born from progenitors located in the ventricular zone (VZ), from where they migrate to assemble into circuits. How neuronal identity is dynamically specified upon progenitor division is unknown. Here, we study this process using a high-temporal-resolution technology allowing fluorescent tagging of isochronic cohorts of newborn VZ cells. By combining this in vivo approach with single-cell transcriptomics in mice, we identify and functionally characterize neuron-specific primordial transcriptional programs as they dynamically unfold. Our results reveal early transcriptional waves that instruct the sequence and pace of neuronal differentiation events, guiding newborn neurons toward their final fate, and contribute to a road map for the reverse engineering of specific classes of cortical neurons from undifferentiated cells.
In the neocortex, higher-order areas are essential to integrate sensory-motor information and have expanded in size during evolution. How higher-order areas are specified, however, remains largely unknown. Here, we show that the migration and distribution of early-born neurons, the Cajal-Retzius cells (CRs), controls the size of higher-order areas in the mouse somatosensory, auditory, and visual cortex. Using live imaging, genetics, and in silico modeling, we show that subtype-specific differences in the onset, speed, and directionality of CR migration determine their differential invasion of the developing cortical surface. CR migration speed is cell autonomously modulated by vesicle-associated membrane protein 3 (VAMP3), a classically non-neuronal mediator of endosomal recycling. Increasing CR migration speed alters their distribution in the developing cerebral cortex and leads to an expansion of postnatal higher-order areas and congruent rewiring of thalamo-cortical input. Our findings thus identify novel roles for neuronal migration and VAMP3-dependent vesicular trafficking in cortical wiring.
The original version of this article omitted two citations. These papers provide a seminal description of retinal waves (Meister et al., 1991) and their effects on retinogeniculate patterning (Penn et al., 1998). These citations have been added, and the article has now been corrected online.
This protocol describes a fluorescence birthdating technique to label, track and isolate isochronic cohorts of newborn cells in the central nervous system in vivo. Injection of carboxyfluorescein esters into the cerebral ventricle allows pulse-labeling of M-phase progenitors in touch with the ventricle and their progeny across the central nervous system, a procedure we termed FlashTag. Labeled cells can be imaged ex vivo or in fixed tissue, targeted for electrophysiological experiments, or isolated using Fluorescence-Activated Cell Sorting (FACS) for cell culture or (single-cell) RNA-sequencing. The dye is retained for several weeks, allowing labeled cells to be identified postnatally. This protocol describes the labeling procedure using in utero injection, the isolation of live cells using FACS, as well as the processing of labeled tissue using immunohistochemistry.intraventricular injection of a carboxyfluorescein ester (a procedure we termed "FlashTag", FT). This procedure allows fluorescent tagging of VZ progenitors and tracking of time-locked cohorts of their postmitotic progeny, including neurons, throughout corticogenesis and early postnatal development.
Birthdating techniques: an overviewThe first-developed and probably still most widely used technique to permanently label neurons based on their embryonic time of birth is 5-bromo-3′-deoxy-uridine (BrdU) birthdating 2,3 . BrdU is a thymidine analogue that is incorporated into cells during DNA synthesis, i.e. during S phase. Administration of BrdU (usually through intraperitoneal (i.p.) injection in pregnant dams or via drinking water) results in systemic labeling of all S-phase
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